Membrane-Mimicking Reverse Micelles for High-Resolution Interfacial Study of Proteins and Membranes

Abstract

Despite substantial advances, the study of proteins interacting with membranes remains a significant challenge. While integral membrane proteins have been a major focus of recent efforts, peripheral membrane proteins (PMPs) and their interactions with membranes and lipids have far less high-resolution information available. Their small size and the dynamic nature of their interactions have stalled detailed interfacial study using structural methods like cryo-EM and X-ray crystallography. A major roadblock for the structural analysis of PMP interactions is limitations in membrane models to study the membrane recruited state. Commonly used membrane mimics such as liposomes, bicelles, nanodiscs, and micelles are either very large or composed of non-biological detergents, limiting their utility for the NMR study of PMPs. While there have been previous successes with integral and peripheral membrane proteins, currently employed reverse micelle (RM) compositions are optimized for their inertness with proteins rather than their ability to mimic membranes. Applying more native, membrane-like lipids and surfactants promises to be a valuable advancement for the study of interfacial interactions between proteins and membranes. Here, we describe the development of phosphocholine-based RM systems that mimic biological membranes and are compatible with high-resolution protein NMR. We demonstrate new formulations that are able to encapsulate the model soluble protein, ubiquitin, with minimal perturbations of the protein structure. Furthermore, one formula, DLPC:DPC, allowed the encapsulation of the PMPs glutathione peroxidase 4 (GPx4) and phosphatidylethanolamine-binding protein 1 (PEBP1) and enabled the embedment of these proteins, matching the expected interactions with biological membranes. Dynamic light scattering and small-angle X-ray scattering characterization of the RMs reveals small, approximately spherical, and non-aggregated particles, a prerequisite for protein NMR and other avenues of study. The formulations presented here represent a new tool for the study of elusive PMP interactions and other membrane interfacial investigations.

Investigating protein‐membrane interactions using native reverse micelles constructed from naturally sourced lipids

“…This corresponds well with a proposed mechanism of fatty acid transfer by FABPs, that the central fatty acid-binding cavity needs to open through localized protein unfolding to transfer fatty acids into or out of the membrane (Lenz et al, 2023;Xiao et al, 2020). Comparison of GPx4 and PEBP1 nRM 15 N-HSQC spectra to aqueous 15 N-HSQCs (Figure 4e, f) reveals interaction of both of these PMPs with the interior surface of the nRMs through their previously characterized membrane binding interfaces (Labrecque et al, 2022;Labrecque & Fuglestad, 2021). PEBP1 has several shifting resonances with CSPs greater than the 20% trimmed mean plus 1σ (0.015 ppm) including those corresponding to L41, Y81, W84-H86, V107-S109, R119, S142, G143, G147, and Y181.…”

“…Encapsulation in PBL and BHL based nRMs show modest shifting of ubiquitin spectral resonances (Figure S5C, D), comparable to shifting observed in SL:DPC nRMs with W 0 = 20 (Figure 3b). This effect was not previously observed for DLPC:DPC mmRMs (Labrecque et al, 2022).…”

“…Residues T9, T22, D32, L43, K48, Q49, E51, L69, and R72-G76 all shift more than a 20% trimmed mean (truncating the highest 20% CSPs only) plus 1σ (0.022 ppm, Figure 3b). Ubiquitin is known to interact weakly with anionic surfaces and these residues match well with those that are known to form the interface (Labrecque et al, 2022;Nucci et al, 2011;Shaw et al, 2011). Throughout these tests, 1-hexanol was titrated into the samples, from an initial concentration of 50 mM, until full sample clarity was achieved.…”

“…Assignments for aqueous ubiquitin were taken from those previously reported (BMRB 25972) (Surana & Das, 2016) and mapped to the spectrum of encapsulated ubiquitin as previously described (Labrecque et al, 2022).…”

“…RM development for protein applications has generally focused on aqueous proteins with minimal perturbations to structure upon encapsulation as a major goal (Dodevski et al, 2014 ; Nucci et al, 2014 ). A recent advance used phosphatidylcholine surfactants to develop membrane‐mimicking reverse micelles (mmRMs) constructed from a mixture of 1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine (DLPC) and n‐ dodecylphosphocholine (DPC) with a 1‐hexanol cosurfactant (Labrecque et al, 2022 ). The resulting assembly in‐part models cellular membranes and encapsulates PMPs in their membrane‐embedded state.…”